Naringenin increased 5-aminolevulinic Acid-mediated Photodynamic Therapy induced Apoptosis in SAS Oral Cancer Cell Line

在口腔癌的治療上，手術切除是最快最有效的方法但也造成顎面的缺損，即使顏面重建後，對於口腔的生理功能及外表的改變確實影響病人的生活品質。5-氨基酮戊酸光動力治療法(ALA-PDT)可用於治療口腔癌一二期的病人，其對於腫瘤細胞具有選擇性並造成癌細胞的凋亡，光動力治療法不像放射線治療會對周圍正常細胞造成傷害而導致纖維化發生，完全治療後不會有疤痕存在，更不會造成顎面的缺損，但由於ALA-PDT需要多次的治療且治療費用過高，因此尋求能增強ALA-PDT的藥物是必須的。 在有血清的環境下，細胞內ALA代謝後的光感物質PpIX會在2-3小時達到最大濃度並無法繼續累加，這被認為是PpIX與細胞運輸蛋白ABCG2作用並排出細胞外由白蛋白接收，所以ALA-PDT的效果受到ABCG2的限制。黃烷酮類的""柚皮素""被報導在K562細胞株中具有抑制ABCG2的功能，並減少抗癌藥物的排出，另有研究也指出柚皮素具有促進癌細胞凋亡的潛力且對正常細胞沒有影響，因此本篇研究結合柚皮素及ALA-PDT利用口腔癌細胞株做研究並探討其合併效果。 研究結果顯示口腔癌細胞株SAS, Ca9-22, HSC3均表現ABCG2蛋白，但柚皮素無法抑制ABCG2蛋白的表現，在功能層面上，加入ALA數個小時，柚皮素及ABCG2中和抗體皆能使SAS細胞內PpIX累積上升，且隨著時間增加能明顯上升。雖然柚皮素幾乎不抑制SAS的細胞活性也不造成細胞凋亡，但ALA-PDT與柚皮素的共同作用下更抑制SAS的細胞活性並增強細胞凋亡，且長時間的ALA處理有更好的效果。同時也發現柚皮素能增加細胞內的1O2，而高濃度的柚皮素也增加細胞內的ROS，在與ALA-PDT的共同作用下更增加1O2, ROS的量，是導致細胞死亡的可能原因。再者，為模擬低氧環境以氯化鈷處理SAS細胞，HIF-1α也因而過度表現，再加入柚皮素後隨著柚皮素濃度上升而HIF-1α表現下降，原本加入氯化鈷而導致的PpIX累積量下降也在加入柚皮素後PpIX回升，並增加光動力治療法的效果。結論，柚皮素可能具有抑制ABCG2的功能使PpIX累積增加並且增加細胞內的1O2及ROS，同時也發現柚皮素能抑制HIF-1α並回復模擬低氧的狀況使PpIX回升，增加模擬低氧狀況下光動力治療法的效果。Surgery is the quickest and most effective way for the oral cancer treatments, but it also causes facial defects and deformity in the surgery region. Even with reconstructive procedures, the patient’s quality of life can be significantly lowered by these functional and facial problems. 5- Aminolevulinic acid photodynamic therapy (ALA-PDT) can be used in the treatment of early oral cancers (stage I and II), and it may cause tumor cell apoptosis selectivity. Unlike radiotherapy, ALA-PDT does not affect the surrounding normal cells. There is no scar formation after treatment. However, due to the need of several ALA-PDT treatments to achieve complete response and high costs, it is necessary to find a drug that can enhance ALA-PDT efficacy. Intracellular photosensitizer PpIX reaches the maximum concentration by 2-3 hours when treated with ALA and it is unable to accumulate PpIX in the presence of serum. It was reported that ABCG2 can transport PpIX to the extracellular albumin, and ABCG2 is believed to act as a regulator of intracellular PpIX levels. Thus, the effect of ALA-PDT is influenced by the ABCG2. Besides, flavanones naringenin has been reported to inhibit ABCG2 function and ABCG2-mediated drug efflux in K562 cell line. Several studies have demonstrated that naringenin have the potential to promote cancer cell apoptosis and had no effect on normal cells. In this study, we investigated the effect of naringenin on ALA-PDT in oral cancer cell line. In the present study, we found that oral cancer cell lines Sas, Ca9-22, Hsc-3 could express ABCG2 protein, but naringenin could not inhibit ABCG2 protein expression. In the functional assay, we blocked ABCG2 by using ABCG2 antibody or naringenin in SAS cell line. We found that the pretreatment of ABCG2 antibody or naringenin could significantly increase the PpIX level in SAS cell line when treated with ALA several hours compared to control. Although naringenin could not inhibit cell viability and induce cell apoptosis, naringenin-pretreated ALA-PDT significantly inhibited cell viability and also enhanced cell apoptosis and It became more effective when treated ALA with a longer period. Moreover, we found that naringenin could increase not only intracellular singlet oxygen but also reactive oxygen species in a high concentration. The naringenin-pretreated ALA-PDT significantly enhanced singlet oxygen and reactive oxygen species production, as a result, it was the possible cause of cell death. Furthermore, in order to simulate a hypoxia environment, SAS cells were treated with cobalt chloride. After a while, HIF-1α protein overexpressed when treated with cobalt chloride. Interestingly, when the naringenin concentration increased, HIF-1α expression decreased significantly. The situation of PpIX accumulation dropped when treated with cobalt chloride, and this could be rescued when treated with naringenin. In summary, naringenin can probably inhibit ABCG2 function that in turn cause more PpIX accumulation and increases intracellular singlet oxygen and ROS. Moreover, naringenin can rescue hypoxia-mimetic conditions and promote PpIX accumulation and further enhances the effect of ALA-PDT